Abstract：
Pathogen infections including Shigella flexneri have posed a significant threat to human health for numerous years. Although culturing and qPCR were the gold standards for pathogen detection, time-consuming and instrument-dependent restrict their application in rapid diagnosis and economically less-developed regions. Thus, it is urgently needed to develop rapid, simple, sensitive, accurate, and low-cost detection methods for pathogen detection. In this study, an immunomagnetic beads-recombinase polymerase amplification-CRISPR/Cas12a (IMB-RPA-CRISPR/Cas12a) method was built based on a cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of S. flexneri in the laboratory. Firstly, S. flexneri was specifically captured and enriched by IMB (Shigella antibody-coated magnetic beads), and the genomic DNA was released and used as the template in the RPA reaction. Then, the RPA products were mixed with the pre-loaded CRISPR/Cas12a for fluorescence visualization. The results were observed by naked eyes under LED blue light, with a sensitivity of 5 CFU/mL in a time of 70 min. With no specialized equipment or complicated technical requirements, the IMB-RPA-CRISPR/Cas12a diagnostic method can be used for visual, rapid, and simple detection of S. flexneri and can be easily adapted to monitoring other pathogens.
摘要：
多年来，包括福氏志贺氏菌在内的病原体感染对人类健康构成了重大威胁。尽管培养和qPCR是病原体检测的金标准，耗时和依赖仪器限制了它们在快速诊断和经济欠发达地区的应用。因此，迫切需要快速发展，简单,敏感,准确,和用于病原体检测的低成本检测方法。在这项研究中,免疫磁珠-重组酶聚合酶扩增-CRISPR/Cas12a（IMB-RPA-CRISPR/Cas12a）方法基于级联信号扩增策略，超敏感,以及在实验室中目测检测福氏链球菌。首先,福氏杆菌被IMB(志贺氏菌抗体包被的磁珠)特异性捕获和富集，基因组DNA被释放并用作RPA反应的模板。然后，将RPA产物与预加载的CRISPR/Cas12a混合用于荧光可视化.结果在LED蓝光下通过肉眼观察,灵敏度为5CFU/mL，时间为70分钟。没有专用设备或复杂的技术要求，IMB-RPA-CRISPR/Cas12a诊断方法可用于视觉，快速,和简单的检测福氏链球菌，可以很容易地适应监测其他病原体。
